The first objective of the proposed research is to determine the complete primary structure of the E. coli cyclic AMP receptor protein, an allosteric protein which exerts a positive control on polycistronic mRNA protein synthesis of enzymes of the lac operon in response to intracellular levels of cAMP. Initial structural data by the prinicipal investigator has resulted in the determination of the N-terminal 14 amino acid sequence of E. coli CRP in successful cleavage, purification and partial characterization of the CNBr and maleoylated tryptic peptides of CRP. Sequence analysis of these peptides will now be undertaken. Additional sequence information on CRP will be obtained by sequence analysis of peptides produced by cleavage at glutamic acid residues and at tryptopan residues. In addition, core fragments of CRP produced by digestion of CRP and cAMP with subtilisin, trypsin, chymotrypsin and S. aureus protease will also be subjected to extended automated N-terminal sequence analysis. Overlapping sequences of all these peptide fragments of CRP will be used to align the CRP amino acid sequence. The second objective of this proposal is to determine the site(s) in the CRP molecule responsible for DNA and cAMP binding. This will be accomplished by studying the structure of mutant strains of bacteria which have altered CRP capacities. These CRP structures will be initially analyzed and compared to wild-type E. coli CRP by reverse phase high performance liquid chromatography and 2-dimensional peptide mapping of peptide fragments produced by enzymatic digestion. Purified peptide fragments will then be subjected to sequence analysis in an attempt to correlate structural changes in CRP with altered cAMP and DNA binding.